A method for measuring the protease activity of c3 and c5 convertase of the alternative complement pathway

Abstract

A method for measuring the protease activity of a convertase of the alternative complement pathway is provided. The method typically comprises immobilizing a biotinylated C3b on a solid phase coated with a biotin binding protein. Substantially homogeneous components of the alternative complement pathway may be incubated with the immobilized C3b in a serum free and gelatin free buffer, to form a convertase. The activity of the convertase is generally measured with an immunoassay.

1 . A method for measuring the protease activity of C3 the steps of:
 a. covalently attaching biotin to C3b to produce biotinylated-C3b;   b. binding biotinylated-C3b to a biotin binding protein immobilized on a solid phase;   c. incubating the immobilized biotinylated-C3b, Factor D, and Factor B in a buffer to form a C3 convertase;   d. transferring C3 to the buffer to cleave C3 with the convertase to form a C3a and C3b; and   e. measuring the amount of C3a with an immunoassay, wherein each of the individual components bio-C3b, Factor B, Factor D, and C3 are substantially homogeneous.   
     
     
         2 . A method for measuring the protease activity of C5 convertase comprising the steps of:
 a. covalently attaching biotin to C3b to produce biotinylated-C3b;   b. binding biotinylated-C3b to a biotin binding protein immobilized on a solid phase;   c. incubating the immobilized biotinylated-C3b, Factor D, and Factor B in a buffer to form C5 convertase;   d. transferring C5 to the buffer, and cleaving C5 with the convertase to form a C5a and CSb; and   e. measuring the amount of the C5a with an immunoassay, wherein each of the individual components bio-C3b, Factor B, Factor D, and C5 are substantially homogeneous.   
     
     
         3 . The method of  claim 1 , wherein the biotin-binding protein is selected from the group consisting of avidin, streptavidin and neutravidin. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein the buffer further comprises properdin and/or is a serum free and gelatin free buffer. 
     
     
         6 . The method of  claim 1 , wherein the convertase comprises a C3b subunit and a Bb subunit in about a 1:1 ratio or a 2:1 ratio. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the homogeneity of each of the components of the pathway is greater than about 90%. 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the solid phase comprises spheres coated with a biotin binding protein. 
     
     
         13 - 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the immunoassay is an ELISA or MSD. 
     
     
         16 . The method of  claim 1 , wherein the immunoassay comprises a step of detecting C3a with an antibody having a high affinity for C3a, and a low affinity for C3. 
     
     
         17 . The method of  claim 16 , wherein the antibody is a neo-epitope antibody. 
     
     
         18 . The method of  claim 16 , wherein the antibody has a Kd from about 10 −6  to about 10 −12  for C3a, and a Kd from about 10 −3  to about 10 −5  for C3. 
     
     
         19 . The method of  claim 2 , wherein the immunoassay comprises a step of detecting C5a with an antibody having a high affinity for C5a, and a low affinity for C5. 
     
     
         20 . The method of  claim 19 , wherein the antibody is a neo-epitope antibody. 
     
     
         21 . The method of  claim 19 , wherein the antibody has a Kd from about 10 −6  to about 10 −12  for C5a, and a Kd from about 10 −3  to about 10 −5  for C5. 
     
     
         22 . (canceled) 
     
     
         23 . A kit for measuring the protease activity of C3 convertase of the alternative pathway using substantially homogeneous components of the alternative complement pathway, the kit comprising:
 a. substantially homogeneous biotinylated C3b;   b. a solid phase coated with a biotin binding protein;   c. substantially homogeneous Factor B, Factor D and C3; and, d. an anti-C3a antibody.   
     
     
         24 - 34 . (canceled) 
     
     
         35 . A kit for measuring the protease activity of C5 convertase of the alternative pathway using substantially homogeneous components of the alternative complement pathway, the kit comprising:
 a. substantially homogeneous biotinylated C3b;   b. a solid phase coated with a biotin binding protein;   c. substantially homogeneous Factor B, Factor D and C5; and,   d. an anti-C5a antibody.   
     
     
         36 - 46 . (canceled) 
     
     
         47 . The method of  claim 2 , wherein the biotin-binding protein is selected from the group consisting of avidin, streptavidin and neutravidin. 
     
     
         48 . The method of  claim 2 , wherein the buffer further comprises properdin and/or is a serum free and gelatin free buffer. 
     
     
         49 . The method of  claim 2 , wherein the convertase comprises a C3b subunit and a Bb subunit in about a 1:1 ratio or a 2:1 ratio. 
     
     
         50 . The method of  claim 2 , wherein the homogeneity of each of the components of the pathway is greater than about 90%. 
     
     
         51 . The method of  claim 2 , wherein the solid phase comprises spheres coated with a biotin binding protein. 
     
     
         52 . The method of  claim 2 , wherein the immunoassay is an ELISA or MSD.